ß1 integrin signaling governs necroptosis via the chromatin-remodeling factor CHD4.

Fibrosis, characterized by sustained activation of myofibroblasts and excessive extracellular matrix (ECM) deposition, is known to be associated with chronic inflammation. Receptor-interacting protein kinase 3 (RIPK3), the central kinase of necroptosis signaling, is upregulated in fibrosis and contributes to tumor necrosis factor (TNF)-mediated inflammation. In bile-duct-ligation-induced liver fibrosis, we found that myofibroblasts are the major cell type expressing RIPK3. Genetic ablation of ß1 integrin, the major profibrotic ECM receptor in fibroblasts, not only abolished ECM fibrillogenesis but also blunted RIPK3 expression via a mechanism mediated by the chromatin-remodeling factor chromodomain helicase DNA-binding protein 4 (CHD4). While the function of CHD4 has been conventionally linked to the nucleosome-remodeling deacetylase (NuRD) and CHD4-ADNP-HP1(ChAHP) complexes, we found that CHD4 potently repressed a set of genes, including Ripk3, with high locus specificity but independent of either the NuRD or the ChAHP complex. Thus, our data uncover that ß1 integrin intrinsically links fibrotic signaling to RIPK3-driven inflammation via a novel mode of action of CHD4.

Genome-wide RNAi screen for regulators of UPRmt in Caenorhabditis elegans mutants with defects in mitochondrial fusion.

Mitochondrial dynamics plays an important role in mitochondrial quality control and the adaptation of metabolic activity in response to environmental changes. The disruption of mitochondrial dynamics has detrimental consequences for mitochondrial and cellular homeostasis and leads to the activation of the mitochondrial unfolded protein response (UPRmt), a quality control mechanism that adjusts cellular metabolism and restores homeostasis. To identify genes involved in the induction of UPRmt in response to a block in mitochondrial fusion, we performed a genome-wide RNAi screen in Caenorhabditis elegans mutants lacking the gene fzo-1, which encodes the ortholog of mammalian Mitofusin, and identified 299 suppressors and 86 enhancers. Approximately 90% of these 385 genes are conserved in humans, and one-third of the conserved genes have been implicated in human disease. Furthermore, many have roles in developmental processes, which suggests that mitochondrial function and their response to stress are defined during development and maintained throughout life. Our dataset primarily contains mitochondrial enhancers and non-mitochondrial suppressors of UPRmt, indicating that the maintenance of mitochondrial homeostasis has evolved as a critical cellular function, which, when disrupted, can be compensated for by many different cellular processes. Analysis of the subsets "non-mitochondrial enhancers" and "mitochondrial suppressors" suggests that organellar contact sites, especially between the ER and mitochondria, are of importance for mitochondrial homeostasis. In addition, we identified several genes involved in IP3 signaling that modulate UPRmt in fzo-1 mutants and found a potential link between pre-mRNA splicing and UPRmt activation.

The Integrated RNA Landscape of Renal Preconditioning against Ischemia-Reperfusion Injury.

BACKGROUND: Although AKI lacks effective therapeutic approaches, preventive strategies using preconditioning protocols, including caloric restriction and hypoxic preconditioning, have been shown to prevent injury in animal models. A better understanding of the molecular mechanisms that underlie the enhanced resistance to AKI conferred by such approaches is needed to facilitate clinical use. We hypothesized that these preconditioning strategies use similar pathways to augment cellular stress resistance. METHODS: To identify genes and pathways shared by caloric restriction and hypoxic preconditioning, we used RNA-sequencing transcriptome profiling to compare the transcriptional response with both modes of preconditioning in mice before and after renal ischemia-reperfusion injury. RESULTS: The gene expression signatures induced by both preconditioning strategies involve distinct common genes and pathways that overlap significantly with the transcriptional changes observed after ischemia-reperfusion injury. These changes primarily affect oxidation-reduction processes and have a major effect on mitochondrial processes. We found that 16 of the genes differentially regulated by both modes of preconditioning were strongly correlated with clinical outcome; most of these genes had not previously been directly linked to AKI. CONCLUSIONS: This comparative analysis of the gene expression signatures in preconditioning strategies shows overlapping patterns in caloric restriction and hypoxic preconditioning, pointing toward common molecular mechanisms. Our analysis identified a limited set of target genes not previously known to be associated with AKI; further study of their potential to provide the basis for novel preventive strategies is warranted. To allow for optimal interactive usability of the data by the kidney research community, we provide an online interface for user-defined interrogation of the gene expression datasets (http://shiny.cecad.uni-koeln.de:3838/IRaP/).

Stage-specific combinations of opposing poly(A) modifying enzymes guide gene expression during early oogenesis.

RNA-modifying enzymes targeting mRNA poly(A) tails are universal regulators of post-transcriptional gene expression programs. Current data suggest that an RNA-binding protein (RBP) directed tug-of-war between tail shortening and re-elongating enzymes operates in the cytoplasm to repress or activate specific mRNA targets. While this concept is widely accepted, it was primarily described in the final meiotic stages of frog oogenesis and relies molecularly on a single class of RBPs, i.e. CPEBs, the deadenylase PARN and cytoplasmic poly(A) polymerase GLD-2. Using the spatial and temporal resolution of female gametogenesis in the nematode C. elegans, we determined the distinct roles of known deadenylases throughout germ cell development and discovered that the Ccr4-Not complex is the main antagonist to GLD-2-mediated mRNA regulation. We find that the Ccr4-Not/GLD-2 balance is critical for essentially all steps of oocyte production and reiteratively employed by various classes of RBPs. Interestingly, its two deadenylase subunits appear to affect mRNAs stage specifically: while a Caf1/GLD-2 antagonism regulates mRNA abundance during all stages of oocyte production, a Ccr4/GLD-2 antagonism regulates oogenesis in an mRNA abundance independent manner. Our combined data suggests that the Ccr4-Not complex represents the evolutionarily conserved molecular opponent to GLD-2 providing an antagonistic framework of gene-specific poly(A)-tail regulation.

Compromised Mitochondrial Protein Import Acts as a Signal for UPRmt.

The induction of the mitochondrial unfolded protein response (UPRmt) results in increased transcription of the gene encoding the mitochondrial chaperone HSP70. We systematically screened the C. elegans genome and identified 171 genes that, when knocked down, induce the expression of an hsp-6 HSP70 reporter and encode mitochondrial proteins. These genes represent many, but not all, mitochondrial processes (e.g., mitochondrial calcium homeostasis and mitophagy are not represented). Knockdown of these genes leads to reduced mitochondrial membrane potential and, hence, decreased protein import into mitochondria. In addition, it induces UPRmt in a manner that is dependent on ATFS-1 but that is not antagonized by the kinase GCN-2. We propose that compromised mitochondrial protein import signals the induction of UPRmt and that the mitochondrial targeting sequence of ATFS-1 functions as a sensor for this signal.

STAT3ß is a tumor suppressor in acute myeloid leukemia.

Signal transducer and activator of transcription 3 (STAT3) exists in 2 alternatively spliced isoforms, STAT3α and STAT3ß. Although truncated STAT3ß was originally postulated to act as a dominant-negative form of STAT3α, it has been shown to have various STAT3α-independent regulatory functions. Recently, STAT3ß gained attention as a powerful antitumorigenic molecule in cancer. Deregulated STAT3 signaling is often found in acute myeloid leukemia (AML); however, the role of STAT3ß in AML remains elusive. Therefore, we analyzed the STAT3ß/α messenger RNA (mRNA) expression ratio in AML patients, where we observed that a higher STAT3ß/α mRNA ratio correlated with a favorable prognosis and increased overall survival. To gain better understanding of the function of STAT3ß in AML, we engineered a transgenic mouse allowing for balanced Stat3ß expression. Transgenic Stat3ß expression resulted in decelerated disease progression and extended survival in PTEN- and MLL-AF9-dependent AML mouse models. Our findings further suggest that the antitumorigenic function of STAT3ß depends on the tumor-intrinsic regulation of a small set of significantly up- and downregulated genes, identified via RNA sequencing. In conclusion, we demonstrate that STAT3ß plays an essential tumor-suppressive role in AML.

Mitochondrial fusion is required for regulation of mitochondrial DNA replication.

Mitochondrial dynamics is an essential physiological process controlling mitochondrial content mixing and mobility to ensure proper function and localization of mitochondria at intracellular sites of high-energy demand. Intriguingly, for yet unknown reasons, severe impairment of mitochondrial fusion drastically affects mtDNA copy number. To decipher the link between mitochondrial dynamics and mtDNA maintenance, we studied mouse embryonic fibroblasts (MEFs) and mouse cardiomyocytes with disruption of mitochondrial fusion. Super-resolution microscopy revealed that loss of outer mitochondrial membrane (OMM) fusion, but not inner mitochondrial membrane (IMM) fusion, leads to nucleoid clustering. Remarkably, fluorescence in situ hybridization (FISH), bromouridine labeling in MEFs and assessment of mitochondrial transcription in tissue homogenates revealed that abolished OMM fusion does not affect transcription. Furthermore, the profound mtDNA depletion in mouse hearts lacking OMM fusion is not caused by defective integrity or increased mutagenesis of mtDNA, but instead we show that mitochondrial fusion is necessary to maintain the stoichiometry of the protein components of the mtDNA replisome. OMM fusion is necessary for proliferating MEFs to recover from mtDNA depletion and for the marked increase of mtDNA copy number during postnatal heart development. Our findings thus link OMM fusion to replication and distribution of mtDNA.

A transcriptomics resource reveals a transcriptional transition during ordered sarcomere morphogenesis in flight muscle.

Muscles organise pseudo-crystalline arrays of actin, myosin and titin filaments to build force-producing sarcomeres. To study sarcomerogenesis, we have generated a transcriptomics resource of developing Drosophila flight muscles and identified 40 distinct expression profile clusters. Strikingly, most sarcomeric components group in two clusters, which are strongly induced after all myofibrils have been assembled, indicating a transcriptional transition during myofibrillogenesis. Following myofibril assembly, many short sarcomeres are added to each myofibril. Subsequently, all sarcomeres mature, reaching 1.5 µm diameter and 3.2 µm length and acquiring stretch-sensitivity. The efficient induction of the transcriptional transition during myofibrillogenesis, including the transcriptional boost of sarcomeric components, requires in part the transcriptional regulator Spalt major. As a consequence of Spalt knock-down, sarcomere maturation is defective and fibers fail to gain stretch-sensitivity. Together, this defines an ordered sarcomere morphogenesis process under precise transcriptional control - a concept that may also apply to vertebrate muscle or heart development.

The complete and fully assembled genome sequence of Aeromonas salmonicida subsp. pectinolytica and its comparative analysis with other Aeromonas species: investigation of the mobilome in environmental and pathogenic strains.

BACKGROUND: Due to the predominant usage of short-read sequencing to date, most bacterial genome sequences reported in the last years remain at the draft level. This precludes certain types of analyses, such as the in-depth analysis of genome plasticity. RESULTS: Here we report the finalized genome sequence of the environmental strain Aeromonas salmonicida subsp. pectinolytica 34mel, for which only a draft genome with 253 contigs is currently available. Successful completion of the transposon-rich genome critically depended on the PacBio long read sequencing technology. Using finalized genome sequences of A. salmonicida subsp. pectinolytica and other Aeromonads, we report the detailed analysis of the transposon composition of these bacterial species. Mobilome evolution is exemplified by a complex transposon, which has shifted from pathogenicity-related to environmental-related gene content in A. salmonicida subsp. pectinolytica 34mel. CONCLUSION: Obtaining the complete, circular genome of A. salmonicida subsp. pectinolytica allowed us to perform an in-depth analysis of its mobilome. We demonstrate the mobilome-dependent evolution of this strain's genetic profile from pathogenic to environmental.

Vav Proteins Are Key Regulators of Card9 Signaling for Innate Antifungal Immunity.

Fungal infections are major causes of morbidity and mortality, especially in immunocompromised individuals. The innate immune system senses fungal pathogens through Syk-coupled C-type lectin receptors (CLRs), which signal through the conserved immune adaptor Card9. Although Card9 is essential for antifungal defense, the mechanisms that couple CLR-proximal events to Card9 control are not well defined. Here, we identify Vav proteins as key activators of the Card9 pathway. Vav1, Vav2, and Vav3 cooperate downstream of Dectin-1, Dectin-2, and Mincle to engage Card9 for NF-κB control and proinflammatory gene transcription. Although Vav family members show functional redundancy, Vav1/2/3-/- mice phenocopy Card9-/- animals with extreme susceptibility to fungi. In this context, Vav3 is the single most important Vav in mice, and a polymorphism in human VAV3 is associated with susceptibility to candidemia in patients. Our results reveal a molecular mechanism for CLR-mediated Card9 regulation that controls innate immunity to fungal infections.

Mechanical regulation of transcription controls Polycomb-mediated gene silencing during lineage commitment.

Tissue mechanics drive morphogenesis, but how forces are sensed and transmitted to control stem cell fate and self-organization remains unclear. We show that a mechanosensory complex of emerin (Emd), non-muscle myosin IIA (NMIIA) and actin controls gene silencing and chromatin compaction, thereby regulating lineage commitment. Force-driven enrichment of Emd at the outer nuclear membrane of epidermal stem cells leads to defective heterochromatin anchoring to the nuclear lamina and a switch from H3K9me2,3 to H3K27me3 occupancy at constitutive heterochromatin. Emd enrichment is accompanied by the recruitment of NMIIA to promote local actin polymerization that reduces nuclear actin levels, resulting in attenuation of transcription and subsequent accumulation of H3K27me3 at facultative heterochromatin. Perturbing this mechanosensory pathway by deleting NMIIA in mouse epidermis leads to attenuated H3K27me3-mediated silencing and precocious lineage commitment, abrogating morphogenesis. Our results reveal how mechanics integrate nuclear architecture and chromatin organization to control lineage commitment and tissue morphogenesis.

The RNA-binding protein Arrest (Bruno) regulates alternative splicing to enable myofibril maturation in Drosophila flight muscle.

In Drosophila, fibrillar flight muscles (IFMs) enable flight, while tubular muscles mediate other body movements. Here, we use RNA-sequencing and isoform-specific reporters to show that spalt major (salm) determines fibrillar muscle physiology by regulating transcription and alternative splicing of a large set of sarcomeric proteins. We identify the RNA-binding protein Arrest (Aret, Bruno) as downstream of salm. Aret shuttles between the cytoplasm and nuclei and is essential for myofibril maturation and sarcomere growth of IFMs. Molecularly, Aret regulates IFM-specific splicing of various salm-dependent sarcomeric targets, including Stretchin and wupA (TnI), and thus maintains muscle fiber integrity. As Aret and its sarcomeric targets are evolutionarily conserved, similar principles may regulate mammalian muscle morphogenesis.

The cytoplasmic poly(A) polymerases GLD-2 and GLD-4 promote general gene expression via distinct mechanisms.

Post-transcriptional gene regulation mechanisms decide on cellular mRNA activities. Essential gatekeepers of post-transcriptional mRNA regulation are broadly conserved mRNA-modifying enzymes, such as cytoplasmic poly(A) polymerases (cytoPAPs). Although these non-canonical nucleotidyltransferases efficiently elongate mRNA poly(A) tails in artificial tethering assays, we still know little about their global impact on poly(A) metabolism and their individual molecular roles in promoting protein production in organisms. Here, we use the animal model Caenorhabditis elegans to investigate the global mechanisms of two germline-enriched cytoPAPs, GLD-2 and GLD-4, by combining polysome profiling with RNA sequencing. Our analyses suggest that GLD-2 activity mediates mRNA stability of many translationally repressed mRNAs. This correlates with a general shortening of long poly(A) tails in gld-2-compromised animals, suggesting that most if not all targets are stabilized via robust GLD-2-mediated polyadenylation. By contrast, only mild polyadenylation defects are found in gld-4-compromised animals and few mRNAs change in abundance. Interestingly, we detect a reduced number of polysomes in gld-4 mutants and GLD-4 protein co-sediments with polysomes, which together suggest that GLD-4 might stimulate or maintain translation directly. Our combined data show that distinct cytoPAPs employ different RNA-regulatory mechanisms to promote gene expression, offering new insights into translational activation of mRNAs.